Novel promoters inducible by dna damaging conditions or agents and uses thereof

ABSTRACT

The present invention relates a method of converting a promoter into a promoter which is inducible upon genotoxic compounds or conditions. The present invention further relates to a method of reducing the basal expression level of promoter which is inducible upon genotoxic compounds or conditions. These methods provides novel nucleotide sequences, vectors and host cells for the expression of proteins under the control of genotoxic conditons or compounds. The novel expression system has wide industrial applications into the field of recombinant protein production but has also clinical applications such as the controlled expression of therapeutic compounds in hypoxic tissues such as tumors.

FIELD OF THE INVENTION

The present invention provides novel nucleotides sequences comprisingpromoter sequences with modified response towards DNA damaging agentsand conditions. The invention also relates to vectors comprising thesenovel sequences and hosts transformed with these vectors. The inventionfurther relates to the use of said modified host cells in therapeuticapplications such as the treatment of cancer. The invention also relatesto the use of modified nucleotide sequences, vectors and host cells forthe recombinant production of proteins.

BACKGROUND OF THE INVENTION

In the search for new therapeutic modalities for cancer, gene therapyhas gained enormous interest over the last years. Many strategies toapply gene therapy have been developed and even more vectors to deliverthe gene of interest have been constructed. However, one of the majorpitfalls of gene therapy is still the lack of specificity of genedelivery. Developing a good gene therapy protocol involves the use of atumour-specific vector system and gene expression limited to the tumouronly. This will result in a high therapeutic index: high local tumourcontrol with low systemic side effects.

Recently, the use of bacteria as tumour-specific protein transfer systemhas gained interest. Attenuated Salmonella (Pawelek, J. M. et al, 1997,Cancer Res. 54:4537-4544., Platt, J., S. Sodi et al, 2000, Eur. J.Cancer 36:2397-2402.), anaerobic Bifidobacterium (Zappe, H. et al, 1988,Appl. Environ. Microbiol. 54:1289-1292.) and apathogenic Clostridium(Fox, M. E. et al, 1996, Gene Ther. 3:173-178., Lambin, P. et al, 1998,Anaerobe 4:183-188; Lemmon, M. J. et al, 1997, Gene Ther. 4:791-765.)have shown to give selective colonisation in tumours without thepresence of vegetative bacteria in the normal tissues (Lambin, refsupra). Moreover, the use of bacteria as protein transfer system is verysafe since treatment can be stopped at any time by addition of theappropriate antibiotic (Theys, J. et al, 2001, FEMS Immunol. Med.Microbiol. 30:37-41.) The anaerobic gram positive bacterium Clostridiumacetobutylicum was genetically engineered to express therapeuticproteins like mouse tumour necrosis factor α (mTNF-α) locally in thetumour under the control of a strong but constitutive promoter. (Theys,J. et al, 1999, Appl. Environ. Microbiol. 65:4295-4300.)

Apart from temporal and spatial expression high levels of a therapeuticprotein are desired. This would solve the problems associated withsystemic administration of therapeutic proteins like TNF-α wherehepatotoxicity and life-threatening hypotension occur as majorside-effects (Old L J. 1985, Science 230:630-636.). Limiting expressionof toxic agents to the tumour cell is extremely important if damage tothe surrounding normal tissues is to be avoided. Anaerobic bacteriaselectively colonise the hypoxic-necrotic areas of solid tumours whichare absent in healthy normal tissues and genetically engineered bacteriawill secrete therapeutic proteins locally in the tumour (Theys, J. etal, 2001, Cancer Gene Ther. 8, 247-297; Theys, J. et al, 1999, Appl.Environ. Microbiol. 65:4295-4300). The use of bacterial host with aradio-inducible promoter would prevent expression in other necrotictissues outside the tumour. In this manner, the combination ofradiotherapy, one of the standard treatment modalities in cancer, andgenetically engineered bacteria as tumour specific protein transfersystem, enables the expression of therapeutic agents locally in thetumour due to both spatial and temporal control of protein expression.

Preferably protein expression would only occur after radiotherapy, sogene expression will be switched on and physicians will know from whattime on the therapeutic protein will be present. Hallahan, D. E. et alin (1995) Nature Med. 1:786-791 describe an adenoviral vector whereinTNF-alpha is positioned under the control of the radiation inducibleEgr-1 promoter.

It was earlier demonstrated that the recA promoter, belonging to theSOS-repair system of bacteria, is induced by radiotherapy, already atthe clinically relevant dose of 2 Gy (Nuyts, S. et al, 2001. AnticancerRes. 21:.857-862; Nuyts, S. et al, 2001, Radiat Res. 155:716-726; Nuyts,S. et al, 2001, Gene Therapy, In press.). A single dose of 2 Gysignificantly increased mTNF-α secretion by recombinant clostridia with44%. Moreover, gene activation could be repeated with a second dose of 2Gy, which makes it promising for clinical use, since in patientsettings, daily fractions of 2 Gy are used (Nuyts, S. et al, 2001 citedsupra)

All genes belonging to the SOS-repair system are activated by thepresence of DNA damage. In non-activated conditions, a repressor calledLexA or DinR (for Bacillus subtilis) binds on a specific operatorsequence called respectively SOS-box (for Gram-negative bacteria) orCheo box for Gram-positive bacteria. In addition to its role inhomologous recombination, RecA functions as a coprotease for the LexAprotein. In a healthy cell, LexA represses the expression of genesencoding DNA repair proteins (SOS genes). Upon injury of DNA, LexAcatalyzes its own digestion, thereby allowing synthesis of necessary SOSproteins. However, LexA can only induce self-catalysis when activated bya ssDNA-RecA filament. A single filament will bind and activate severalLexA proteins, each of which then cleaves other bound proteins. Thus,ssDNA-RecA, a product of DNA injury, stimulates DNA repair. through anincreased transcription of the SOS-genes (Cheo, D. L. et al, 1991, J.Bacteriol. 173:1696-1703.; Miller, R., and T. Kokjohn, 1990, Annu. Rev.Microbiol. 44:365-394.). These genes will play a role in repairing theoriginal DNA damage.

Both LexA and DinR bind to their operator sequence as dimers (Kim, B.,and J. W. Little. 1992, Science 255:203-205., Yazawa, K. et al, 2000,Cancer Gene Ther. 7:269-274.). The consensus sequence for the Cheo boxin Gram-positive bacteria is 5′ GAACNNNNGTTC 3′ (cheo et al citedsupra). This consensus sequence is positioned within promoter regionssuch that the regulatory molecule LexA bound at these sites couldinterfere with the initiation of transcription by RNA polymerase.Several genes can be found which have 2 or more putative Cheo boxes andfor those, in which repressor binding is proven, the distance betweenthe two boxes is 15 to 16 bp (yazawa cited supra ).

A system similar as described for Clostridium is known for gram negativebacteria such as E. coli. When cells like E. coli are subject toexcessive DNA damage, a system (the SOS response) that stops DNAsynthesis and invokes massive DNA repair is triggered. The SOS system isregulated by RecA. If there is any DNA damage present duringreplication, RecA will associate with the single stranded DNA that isgenerated after DNA damage. RecA will also associate with a proteincalled LexA. LexA is a repressor that normally turns off a large groupof genes associated with DNA repair, including recA, uvrA, uvrB anduvrD. Each of these genes has a similar consensus sequence called theSOS BOX (5′-CTGNNNNNNNNNNCAG-3′, where N can be any base). LexA binds tothe SOS box, turning off genes with an SOS box in their promoters.However, when RecA interacts with single stranded DNA, RecA is“activated” such that RecA binds to LexA. LexA bound to RecA does notbind to the SOS box, and thus all the genes with an SOS box (mainly DNArepair genes) are turned on. The controlling factor in this system isthe presence of single stranded DNA. Some genes with SOS boxes inhibitcell division. Thus when the LexA-RecA complex is formed, DNA repair isinitiated and cell division is inhibited. When the damaged DNA isrepaired, there will be no means to activate the RecA such that it bindsto LexA, and thus LexA will again inhibit all genes with SOS boxes andrelated DNA repair will cease and cell division will continue.

Despite the wide knowledge on DNA damage mediated expression of proteinsand despite the variety of expression systems for proteins by anaerobicorganisms in hypoxic tissues, there is still a need for DNA constructs,vectors and host cells which allow inducible expression with low levelsof basal expression. There is also a need for DNA constructs, vectorsand host cells which allow more regulated and higher expression ofproteins than those known in the art.

SUMMARY OF THE INVENTION

The present invention relates to an isolated and purified polynucleotidecomprising at least one first sequence element inserted in a secondsequence element wherein the first sequence element is a repressorbinding element of a promoter which is inducible by DNA damaging agentsor conditions and wherein the second sequence element is a promotersequence. The promoter can be not inducible by a DNA damaging agent orcondition but also can be inducible by a DNA damaging agent orcondition. The polynucleotide can be positioned 5′ to a nucleotidesequence suitable for the introduction of a third sequence element.

The invention relates to a method of converting a promoter which is notinducible by DNA damaging agents or conditions into a promoter which isinducible by radiation, genotoxic compounds or DNA damaging compoundscomprising the step of inserting at least one repressor binding elementof a promoter which is inducible by a DNA damaging compound or conditioninto said non inducible promoter.

The invention relates to a method of increasing the induction level of afirst promoter which is inducible by genotoxic compounds or conditionscomprising the step ofinserting at least one repressor binding elementof a said first promoter or a second promoter which is inducible by aDNA damaging compound or condition into the first inducible promoter.

The invention relates to a method of decreasing the basal expressionlevel of a first promoter which is inducible by genotoxic compounds orconditions comprising the step of inserting at least one repressorbinding element of a said first promoter or a second promoter which isinducible by a DNA damaging compound or condition into the firstinducible promoter.

The invention further relates to a vector comprising a nucleotidesequence of the present invention.

The invention further relates to a bacterial host cell transfected withthe vectors of the present invention.

The invention further relates to a pharmaceutical composition comprisinga cell of the present invention in admixture with at least onepharmaceutically acceptable carrier.

The invention further relates to a method of expressing a therapeuticprotein or a protein converting a precursor into a therapeutic compoundcomprising a first step of administering to an individual of thepharmaceutical composition and a second step of subjecting the person toa DNA damaging condition and/or administering to an individual a DNAdamaging compound or a precursor thereof.

The invention also relates to a method for the in vitro production ofrecombinant proteins comprising the step of contacting a culture of hostcells of the present invention with a DNA damaging compound orcondition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the releative increase of mTNF-α secretion in Clostridiumacetobutylicum DSM792 pIMP-recA-mTNF-α without Cheo box (filled boxes)and Clostridium acetobutylicum DSM792 pIMP-recA-mTNF-α with an extraCheo box (empty boxes) after a single dose of 2 Gy in function of timeafter irradiation. The bars represent data from three independentexperiments.

FIG. 2 shows the relative increase of mTNF-α secretion in Clostridiumacetobutylicum DSM792 pIMP-eglA-mTNF-α (filled boxes) and Clostridiumacetobutylicum DSM792 pIMP-eglA-mTNF-α with a Cheo box (empty boxes)after a single dose of 2 Gy in function of time after irradiation. Thebars represent data from three independent experiments.

FIG. 3 presents the results of RT-PCR on irradiated and non-irradiatedRNA extracted from Clostridium acetobutylicum DSM792. The upper panelrepresents the amplification of a 650 bp internal fragment of 16S rRNAwhich functions as an internal standard to ensure equal amounts of RNAwere used in each reverse transcription reaction. The lower panelrepresents the amplification of a 470 bp internal fragment of mTNF-α.Areference DNA-ladder is shown on the right.RNA extracted from C.acetobutylicum DSM792 transformed with pIMP-eglACheo-mTNF-α is shown inlanes 1 and 2; pIMP-recA-mTNF-α is shown in lanes 3 and 4;pIMP-recAextraCheo-mTNF-α is shown in lanes 5 and 6; pIMP-eglA-mTNF-α isshown in lanes 8 and 9; pIMP-recAdeletedCheo-mTNF-α is shown in lanes 10and 11; Lane 7 shows a positive control for 16S rRNA (PCR performed onchromosomal DNA C. acetobutylicum)

FIG. 4 shows the activity of an expressed luciferase reporter geneoperably linked to a recA promoter after (E=exposed; squares) or withoutirradiation (NE=non exposed, diamonds) (RLU=relative light units)

FIG. 5 shows the induction factor of a luciferase gene operably linkedto a RecA promotor. The induction factor is the ratio between theexpression level under inducing conditions and the expression levelunder non inducing conditions.

DEFINITIONS

“consensus sequence” in the present invention refers to a representationof a sequence alignment of related sequences of repressor bindingelements wherein in this representation the most frequently occurringresidue at a certain position is shown. Therefore, the consensussequence represents the consensus sequence and also the naturallyoccurring variations on the consensus sequence as represented by theindividual sequences of the alignment, and also engineered sequenceswhere one or more residues are modified with respect to the consensussequence, said engineered sequence still being able to bind therepressor.

“Expression” as used herein, refers to the transcription and translationto gene product from a polynucleotide and/or a full-length gene codingfor the sequence of the gene product. In the expression, a DNA chaincoding for the sequence of a gene product is first transcribed to acomplementary RNA which is often a messenger RNA and, then, the thustranscribed messenger RNA is translated into the above-mentioned geneproduct if the gene product is a protein.

“Gene products” as used herein, refers to any molecule capable of beingencoded by a nucleic acid, but not limited to, a polypeptide or anothernucleic acid, e.g. DNA, RNA, dsRNA, ribozyme, DNAzyme etc. The term“Gene product” may thus refer to a polypeptide produced by transcriptionof a specific DNA coding region into mRNA followed by translation of themRNA by a ribosome. Such a polypeptide may also refer to as a “protein”.The polynucleotide, which encodes for the gene product of interest, isnot limited to naturally occurring full-length “gene” having non-codingregulatory elements.

“promoter” and “promoter region” as used herein, refer to a sequence ofDNA, usually upstream of the gene product coding sequence, whichcontrols the expression of the coding region by providing therecognition for RNA polymerase and/or other factors required fortranscription to start at the correct site. Promoter sequences arenecessary but not always sufficient to drive the expression of the gen.

The term “strong promoter” as used herein refers to a promoter operablylinked to an encoding polynucleotide sequence that results in highlevels of expression and/or expression independent of cell cycle.

“DNA damaging compound (synonym: genotoxic compound) or DNA damagingagent” as used herein refers to molecules which damage or modify thebackbone/and or side chains resulting in the generation of singlestranded DNA fragments.

“DNA damaging conditions” as used herein relate to conditions such ashigh energy radiation, e.g. UV radiation, gamma or X ray radiation whichmodify or damage the backbone/and or side chains of DNA resulting in thegeneration of single stranded DNA fragments.

“Gram negative” refers to the inability of a bacterium to resistdecolorisation with alcohol after being treated with Gram crystalviolet. Optionally, these bacteria can be counterstained with safranin,imparting a pink or red colour to the bacterium when viewed by lightmicroscopy

“Gram positive” refers to ability of a bacterium to resistdecolorisation with alcohol after being treated with Gram crystalviolet, imparting a violet colour to the bacterium when viewed by lightmicroscopy.

The term “strong promoter inducible by DNA damaging conditions orcompounds” used herein refers to naturally occurring promoters such asthe recA promoter. It also refers to strong constitutive promoters orpromoters that in their nature are not inducible by radiation, bygenotoxic compounds or by DNA damaging agents, but which after insertionof one or more repressor binding elements in said promoter region havebeen made inducible by radiation, by genotoxic compounds or by DNAdamaging agents.

“Inducible promoter” as used herein, refers to a promoter which can beactivated by addition of a particular molecule or a particular agent orby exposing to physical conditions such as irradiation, called aninducer.

“Operably linked” as used herein, refers to a state of joinder of apromoter and a full length gene or an encoding polynucleotide, whereinRNA polymerases are capable of recognising the promoter.

“repressor-binding element” as used herein refers a specific operatorsequence in the promoter sequence of gram positive or gram negativebacteria. For example, repressor binding elements of gram-positivebacteria to which the repressor dinR binds upon DNA damage are sequenceswith the Cheo box consensus sequence GAACNNNNGTTC [SEQ ID NO 1] or theDinR box consensus sequences CGAACRNRYGTTYC [SEQ ID NO 2] Examples ofsuch sequences are shown in table 1. For example, repressor bindingelements of gram-negative bacteria to which the repressor LexA bindsupon DNA damage are sequences with the SOS box consensus sequenceCTGNNNNNNNNNNCAG [SEQ ID NO 3]. Examples of such sequences are shown intable 2.

“therapeutic protein” as used herein relates to any protein used in thetreatment of a mammalian disease. Where the disease involves pathogenictissues such as cancer tumours or bacterial infection, this term canrefer to proteins which have a toxic, cytotoxic or cytostatic effect orcan refer to proteins which convert a molecule into a molecule withcytostatic or cytotoxic effects (e.g. prodrug). Therapeutic proteins asused herein related to the treatment of an ischemic disease can alsorelate to growth factors, angiogenic or arteriogenic factors (e.g. PIGFor VEGF or other fam0ily members). Therapeutic proteins as used hereincan relate to drug delivery can refer to a drug or a pore openingprotein, for example.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising finding that theintroduction of a repressor binding elements such as the Cheo box in aconstitutive promoter such as the eglA promoter causes the modifiedpromoter to respond to ionising radiation in contrast to the unmodifiedeglA promoter. The Cheo box was introduced about 70 bp upstream of theribosome binding site to make sure interaction could occur between RNApolymerase and the promoter which could inhibit transcription. Thispositioning is within the range of between about −42 and about −106reviewed by Yazawa et al (cited supra).

The present invention is further based on the finding that a repressorelement such as the Cheo box in a promoter such as the recA promoter ofa C.acetobutylicum DSM792 is a necessary and sufficient elementresponsible for induction after action of a DNA damaging condition orcompound, e.g. ionising irradiation. After deletion of the Cheo boxthere was no increase in protein expression after irradiation incontrast with the ‘wild-type’ recA promoter where radiation-inducedexpression was present. Incorporation of a second Cheo box of about 50bp upstream of the first, increased radio induced expression from abouta 40% the expressed protein for the ‘wild-type’ promoter to about 400%for the mutated promoter, when compared to the non-irradiatedconditions.

A first embodiment of the invention refers to the insertion of one ormore sequence elements (first sequence elements) into nucleotidesequences of promoters (second sequence elements) which are notinducible by DNA damaging agents or conditions, said insertion orinsertions leading to a modified promoter which becomes inducible uponaction of a DNA damaging agent or condition.

A second embodiment of the invention refers to the insertion of one ormore sequence elements (first sequence elements) into nucleotidesequences of promoters (second sequence elements) which are alreadyinducible by DNA damaging agents or conditions, said insertion orinsertions leading to a modified promoter which remains inducible uponDNA damaging agents or conditions but results in an increased expressionlevel of protein compared to the expression level of the promoter beforethe insertion of one or more first sequence elements.

In one aspect of this embodiment the one or more sequence elements beingintroduced are repressor binding elements of promoters of genes whichare switched on in the presence of DNA damaging compounds or conditions.These genes are mostly involved in the DNA damaging repair processes ofliving cells and occur in a wide range of organisms including highereukaryotes such mammals and vascular plants, but also lower eukaryotessuch as insects and nematodes and fungi, such as fission and buddingyeast and in prokaryotes, including both gram positive and gram negativebacteria. Especially lower organism which live in high environmentalstress (such as light, radiation, and toxic environments) are adapted torespond to these stress conditions by a sophisticated DNA repair system.Promoters which are inducible after DNA damaging conditions or compoundsare well characterised in both gram negative and gram positive bacteria.

In one aspect of the invention, the repressor binding element which isintroduced into a promoter can be any element when said repressorbinding element is being recognised by a repressor in the host in whichthe modified promoter was introduced. The recognition can be evaluatedby contacting the repressor with a putative repressor bindingoligonucleotide. This binding can be assayed for example with a gelretardation assay, also known as “bandshift”, wherein a labellednucleotide shows a retarded migration to a gel when a protein is boundto said labelled nucleotide.

In a more preferred aspect of this embodiment the repressor bindingelements are bacterial repressor binding elements of promoters activatedby DNA damaging conditions or compounds. These are known in grampositive bacteria as Cheo boxes and DinR boxes. The different sequencesof Cheo boxes are represented by the Cheo box consensus site of 12nucleotides GAACNNNNGTTC [SEQ ID NO 1] or alternatively by the DinRconsensus site of 14 nucleotides CGAACRNRYGTTYC [SEQ ID NO 2]. A list ofexamples of repressor binding elements comprising Cheo boxes, withoutthe intention to restrict the scope of the invention only to theseexamples, is shown in Table 1. TABLE 1 Repressor binding sequences ofRecA like sequences from different gram positive bacteria sequencerepressor species promoter binding element SEQ ID number Bacillus rec A(−51) CGAATATGCGTTCG SEQ ID NO 4 subtilis dinA CGAACTTTAGTTCG SEQ ID NO5 dinB AGAACTCATGTTCG SEQ ID NO 6 dinC (−24) CGAACGTATGTTTG SEQ ID NO 7dinC (−53) AGAACAAGTGTTCG SEQ ID NO 8 dinR (−39) CGAACCTATGTTTG SEQ IDNO 9 dinR (−67) CGAACAAACGTTTC SEQ ID NO 10 dinR (−104) GGAATGTTTGTTCGSEQ ID NO 11 Bacteroides recA (−20) CGAATTAAACTTTG SEQ ID NO 12 fragilisrecA (−107) CGAACGGATCATCG SEQ ID NO 13 Clostridium recA AGAACTTATGTTCGSEQ ID NO 14 perfringens Corynebacterium recA CGTAGGAATTTTCG SEQ ID NO15 glutamicum Corynebacterium recA AGAATGGTCGTTAG SEQ ID NO 16pseudotuberculosis Deinococcus recA CGATCCTGCGTAAG SEQ ID NO 17radiodurans Mycobacterium recA CGAACAGATGTTCG SEQ ID NO 18 lepraeMycobacterium recA CGAACAGGTGTTCG SEQ ID NO 19 smegmatis MycobacteriumrecA TCGAACAGGTGTTCGA SEQ ID NO 20 tuberculosis lexA TCGAACACATGTTTGASEQ ID NO 21 Staphylococcus recA CGAACAAATATTCG SEQ ID NO 22 aureusStreptococcus recA CGAACATGCCCTTG SEQ ID NO 23 mutans Streptomyces recACGAACATCCATTCT SEQ ID NO 24 lividans Thermotoga recA CGAATGTCAGTTTG SEQID NO 25 maritima

In another more preferred aspect of this invention the repressor bindingelements are from gram negative bacteria. In these organism the elementis known as the SOS box. The different sequences of SOS boxes arerepresented by the SOS box consensus site of 16 nucleotidesCTGNNNNNNNNNNCAG [SEQ ID NO 3]. A list of examples representing SOSboxes, without the intention to restrict the scope of the invention onlyto these examples, is shown in Table 2. TABLE 2 sos boxes of promotersfrom the gram negative bacterium E. coli. sos sos box sequence box nameSEQ ID tactgtatgagcatacagta recA SEQ ID NO 26 tactgtatattcattcaggt uvrASEQ ID NO 27 aactgtttttttatccagta uvrB SEQ ID NO 28 tactgtacatccatacagtasulA SEQ ID NO 29 atctgtatatatacccagct uvrD SEQ ID NO 30tactgtataaataaacagtt mucAB SEQ ID NO 31 tactgtgtatatatacagta clo13 SEQID NO 32 tgctgtatatactcacagca lexA-1 SEQ ID NO 33 aactgtatatacacccaggglexA-2 SEQ ID NO 34 tgctgtatataaaaccagtg cle1-1 SEQ ID NO 35cagtggttatatgtacagta cle1-2 SEQ ID NO 36 tactgtatatgtatccatat Co11b SEQID NO 37 tactgtatataaacacatgt Co1A-1 SEQ ID NO 38 acatgtgaatatatacagttCo1A-2 SEQ ID NO 39 atctgtacataaaaccagtg Co1E2 SEQ ID NO 40tactgtatataaaaacagta umuDC SEQ ID NO 41 tactgtatataaaaccagtt recN-1 SEQID NO 42 tactgtacacaataacagta recN-2 SEQ ID NO 43

In one embodiment the insertion of one or more repressor bindingelements occurs between 1 and 1000 basepairs upstream from the ribosomebinding element of the second sequence element, alternatively occursbetween 1 and 200 base pairs, or occurs between 46 and 104 basepairsupstream from said ribosome binding element.

Another aspect of this embodiment relates to non inducible promoterswhich are modified by the insertion of one or more repressor bindingelements. These promoters can be weak promoters but are preferablystrong promoters. A candidate promoter can be evaluated by any reporterassay wherein a promoter is operably linked to a reported gene andwhere, after introduction in the appropriate host, the amount oractivity of the reporter gene is assayed. Examples of such reportergenes or luciferase or chloramphenicol transferase. A preferred promoteraccording the present invention is the EglA promoter of Clostridium.

Another aspect of the invention relates to inducible promoters which aremodified by the insertion of one or more repressor binding elements.These promoters can be any promoter which is inducible by DNA damagingcompounds or conditions. Examples of such promoters in gram negativebacteria are promoters to which the repressor LexA can bind and arementioned in table 2. Examples of such promoters in gram positivebacteria are promoters to which the repressor DinR can bind arementioned in table 1.

Another aspect of the present invention are vectors comprising themodified sequences which are inducible upon DNA damaging compound orconditions. Said vectors contain a nucleotide sequence located 5′ to thepromoter (known as cloning site or multiple cloning site) for operablylinking to the promoter a sequence which is described according to thisinvention as a third sequence element. This third sequence element isthe gene which will be transcribed and translated after action by DNAdamaging conditions or compounds. Further, vectors can optionallycomprise an additional nucleotide sequence which results in theexpression of a fusion protein with as a first part of the fusionprotein the protein being expressed from the third sequence element andas a second part a signal peptide, a tag for the recognition of anantibody (e.g. myc, Flag, HA tag), a tag for the recognition of aprotease (e.g. thrombin, Factor X, Enterokinase site), a protein withenzymatic activity or a protein or peptide which is able to bind to acarrier (e.g. GST, maltose binding protein, His tag). A preferredversion of fusion protein is a protein fused to a peptide which allowsthe secretion of the protein. Preferred examples of such signal peptides(and accompanying promoter) are for Clostridium the eglA isolated fromClostridium acetobutylicum, clostripain promoter and signal peptide fromClostridium histolyticum, glutamine synthethase from Clostridiumbeijerinckii

Another aspect of this invention relates to host cells transformed withthe vectors of the present invention. These host cells are preferablybacteria. The bacteria are preferably bacteria which have a certaintissue or organ specific preference.

In the embodiments related to the treatment of an hypoxic tissue, thebacteria preferably are non pathogenic bacteria and even more preferablyanaerobic or facultative anaerobic non pathogenic bacteria; in a mostpreferable embodiment they are anaerobic non pathogenic bacteria; in apreferred embodiment these non pathogenic gram positive anaerobicbacteria are bacteria of the species Clostridium. Examples ofgram-positive bacteria thereof are Clostridium acetobutylicum,Clostridium sporogenes, Clostridium beijerinckii, Clostridiumoncolyticum, Clostridium butyricum, Clostridium novyi as well asBifidobacterium infantis, Bifidobacterium bifidum, Bifidobacteriumlongum

Other examples of Gram-positive bacteria useful for differentapplications are examples of aerobic or facultative aerobic grampositive bacteria (for in vitro expression) Bacillus subtilis,Streptomyces lividans, Streptomyces coelocolor, Lactobacillus spp.,Lactococcus spp.

Examples of gram negative bacteria are Salmonella typhimurium, but alsoother species such as other intracellular bacteria or species such as E.coli, Pseudomonas, Rhizobium.

Another aspect of this invention relates to pharmaceutical compositionsand methods of treating patients with bacterial host cells transfectedwith the nucleotides of the present invention. Preferably, the treatmentaccording to the present invention relates to the treatment of cancertissue and more preferably to the treatment of cancer tissue subject tohypoxic conditions. The treatment however also relates to the treatmentof other disorders with hypoxic conditions such as abscesses andischemic tissues. Recently, the use of bacteria as tumour-specificprotein transfer system has gained interest. Attenuated Salmonella(Pawelek cited supra, Platt cited supra.), anaerobic Bifidobacterium(Zappe et al cited supra) and apathogenic Clostridium (Fox et al, citedsupra, Lambin et al, 1998 cited supra; Lemmon et al cited supra) haveshown to give selective colonisation in tumours without the presence ofvegetative bacteria in the normal tissues (Lambin, ref supra).

The treatment according to the present invention has the advantage thatthe action of irradiation or a administration of a genotoxic compound,in addition to its curative effect, acts as an inducer of a gene whichexpresses a therapeutic protein. As an alternative, genotoxic drugs canbe administered at a lower dosage which results in limited systemic sideeffects. The genotoxic compound will, however, act locally in the hostcell as an inducer of the gene encoding a therapeutic protein.

The treatment of tumours according to the present invention relates totumours occurring in mammals and in particular to humans. Examples oftumours are sarcomas, carcinomas, or other solid tumor cancers include,but are not limited to, germ line tumors, tumors of the central nervoussystem, breast cancer, prostate cancer, cervical cancer, renal cancer,bladder cancer, uterine cancer, lung cancer, ovarian cancer, testicularcancer, thyroid cancer, mesoendothelioma, mesothelioma, astrocytoma,glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer,and melanoma.

In accordance with the present invention the treatment comprisessubjecting an individual to host cells of the present invention, thehost cells having a vector comprising inducible promoter, inducible uponaction of a DNA damaging condition or compound. The DNA damagingcondition can be irradiation with high energy radiation such as betarays, gamma rays or X-rays. The treatment may comprise a single dose ofirradiation or may comprise several doses of irradiation (fractionateddoses). The effective dose of irradiation can be calculated usingmethods known in the art taking into account the overall health of thepatient and the type and location of the solid tumour. An illustrativeexample of a course of radiation treatment for a human patient with asolid tumour is local administration of irradiation to the tumour siteof 2 Gy/day for 5 days per week for 6 weeks (total exposure of 60 Gy).

Alternatively for the treatment of tissues which are located at theoutside of the body or which are easily accessible, treatment withultraviolet radiation or string light sources can be used.

The vectors and the host cells of the present invention allow theinduction of a gene by a compound with genotoxic properties which isbeing used as such in cancer chemotherapy or in a combination treatmentof chemotherapy and irradiation. Examples of such compounds withgenotoxic properties are mitomycin, alkylating agents, antimetabolites,bioreductive drugs.

The present invention allows the inducible expression of genes at aspecific part of the body. The specific part of the body may bedetermined by a tissue state such as hypoxia, or by a particularpreference for a part of the body by the host bacterial cell. Inaccordance with the definitions of the present invention the genes beingencoded are transcribed and translated from what is described as thethird sequence element. This allows the use of genes which are normallytoxic for healthy cells when administered to an individual, and allowsthe use of genes which are capable of converting locally a harmlessprecursor compound into a toxic compound, thereby resulting into thetime and place dependent activity of a anticancer agent. Proteins withtoxic or cytotoxic activities and protein which convert a non toxiccompound (prodrug converting enzymes) into a toxic compound are definedin the context of cancer treatment as therapeutic proteins.

Examples of cytotoxic proteins are saporin, ricins, abrin and ribosomeinactiviting proteins (RIPs), Pseudomonas exotoxin, inhibitors of DNA,RNA or protein synthesis, DNA or RNA cleaving molecules such as DNaseand ribonuclease, proteases, lipases, phospholipase), prodrug convertingenzymes (e. g., thymidine kinase from HSV and bacterial cytosinedeaminase), light-activated porphyrin, ricin, ricin A chain, maize RIP,gelonin, E. coli cytotoxic necrotic factor-1, Vibrio fischeri cytotoxicnecrotic factor-1, cytotoxic necrotic factor-2, Pasteurella multicidatoxin (PMT), cytolethal distending toxin, hemolysin, verotoxin,diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin,pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP),Dianthins 32 and 30, abrin, monodrin, bryodin, shiga, a catalyticinhibitor of protein biosynthesis from cucumber seeds (see, e. g.,International Publication WO 93/24620), Pseudomonas exotoxin, E. coliheat-labile toxin, E. coli heat-stable toxin, EaggEC stable toxin-1(EAST), biologically active fragments of cytotoxins and others known tothose of skill in the art. See, e. g. O'Brian and Holmes, Neidhardt etal. (eds.), pp. 2788-2802, ASMPress, Washington, D. C.) Yet otherexemplary gene products of interest include, but are not limited to,methionase, aspariginase and glycosidase.

Other therapeutic proteins can be antiangiogenic factors, such asendostatin; angiostatin; apomigren; anti-angiogenic antithrombin III;proteolytic fragments of fibronectin; uPA receptor antagonist; I6 kDaproteolytic fragment of prolactin; t 7.8 kDa proteolytic fragment ofplatelet factor-4; anti-angiogenic 13 amino acid fragment of plateletfactor-4; antiangiogenic 14 amino acid fragment of collagen I;anti-angiogenic 19 amino acid peptide fragment of Thrombospondin I;anti-angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGRcontaining peptides; small anti-angiogenic peptides of laminin,fibronectin, procollagen and EGF, and peptide antagonists of integrin av3 and the VEGF receptor; can also be a Flt-3 ligand.

Other therapeutic proteins in accordance with the present invention arecytokines which result in a significant antitumor immune response.Examples are IL-1; IL-2; IL4; IL-5; IL-15; IL-18; IL-12; IL-10; GM-CSF;INF-y; INF-a; SLC; EMAP2; MIP-3a; MIP-3; an MHC gene such as HLA-B7;members of the TNF family, including but not limited to tumor necrosisfactor-a (TNF-a), tumor necrosis factor-P (TNF-P), (TRAIL), (TRANCE);CD40 ligand (CD40L); LT-a; LT-P; OX40L; CD40L; FasL; CD27L; CD30L;4-1BBL; APRIL; LIGHT; TL1; TNFSF16, TNFSF17, and AITR-L.

Examples of therapeutic pro-drug converting enzymes are HSVTK (herpessimplex virus thymidine kinase) and VZVTK (varicella zoster virusthymidine kinase), which selectively phosphorylate certain purinearabinosides and substituted pyrimidine compounds, converting thesecompounds to metabolites that are cytotoxic or cytostatic. For example,exposure of the drug ganciclovir, acyclovir, or any of their analogues(e. g., FIAU, FIAC, DHPG) to cells expressing HSVTK allows conversion ofthe drug into its corresponding active nucleotide triphosphate form; E.coli guanine phosphoribosyl transferase, converting thioxanthine intotoxic thioxanthine monophosphate; alkaline phosphatase, convertinginactive phosphorylated compounds such as mitomycin phosphate anddoxorubicin-phosphate to toxic dephosphorylated compounds; fungal (egFusarium oxysporum) or bacterial cytosine deaminase, which converts5-fluorocytosine to the toxic compound 5-fluorouracil; carboxypeptidaseG2, cleaving glutamic acid from para-N-bis (2-chloroethyl) aminobenzoylglutamic acid, thereby creating a toxic benzoic acid mustard;Penicillin-V amidase, which converts phenoxyacetabide derivatives ofdoxorubicin and melphalan to toxic compounds Moreover, a wide variety ofHerpesviridae thymidine kinases, including both primate and non-primateherpesviruses, are suitable, including Herpes Simplex Virus Type 1Herpes Simplex Virus Type 2 Varicella Zoster Virus.

Other therapeutic compounds are bacterial proteins, which uponexpression in mammalian cells perform a cytotoxic function. Exampleshereof are colicin, such as colicin E3 , V, A, E1, E2, Ia, Ib, K, L, ;cloacin, such as cloacin DF13; pesticin A1122; staphylococcin 1580;butyricin 7423; vibriocin pyocin RI or AP41; megacin A-216 and BRP(Bacteriocin Release Protein) from Enterococus cloacae.

Other therapeutic proteins for use according to the present inventionare pore opening compounds such as the bacterial toxin Zot from Vibriocholerae, which act as pore forming molecules, thereby facilitating thetransport of pharmaceutical small molecules which are normally not ableto penetrate the membrane of a mammalian or human cell.

Other therapeutic proteins according the present inventions arevascularisation promoters or growth factors with angiogenic and/orarteriogenic properties such as members of the VEGF family (PLGF, VEGF)for the treatment of ischemic disease.

In another embodiment of the present invention, a transformed bacterium,preferably with no or little toxic side effects, and preferably with atissue or organ specific preference, is introduced into a patientwherein a persistent bacterial infection occurs (eg rectum, bladder,intestine, stomach). Upon induction with a genotoxic compound or a DNAdamaging condition the host bacterium will express a toxic protein orconverting a pro-drug in to drug toxic compound resulting in the killingof the persistent bacteria. The introduced host cell can, if desired, bekilled with an appropriate antibiotic.

Several methods are known to deliver the bacterial host cell to anindividual being treated according to this invention. Methods ofintroduction include but are not limited to intradermal, intrathecal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e. g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. In a specific embodiment, it may be desirable toadminister the pharmaceutical compositions of the invention locally tothe area in need of treatment; this may be achieved by, for example, andnot by way of limitation, local infusion during surgery, by injection,by means of a catheter, or by means of an implant, said implant being ofa porous, non-porous, or gelatinous material, including membranes, suchas sialastic membranes, or fibers. In one embodiment, administration canbe by direct injection at the site (or former site) of a malignant tumoror neoplastic or pre-neoplastic tissue.

In another embodiment, the host cell of the present invention can bedelivered in a controlled release system. In one embodiment, a pump maybe used. In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974) In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,thus requiring only a fraction of the systemic dose. Other controlledrelease systems are discussed in the review by Langer (1990) Science249, 1527-1533

The pharmaceutical compositions of the present invention comprise a hostcell and at least one pharmaceutically acceptable carrier. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration. In a preferredembodiment, the composition is formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wherethe composition is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

Another embodiment of the present invention is the use of the modifiednucleotide sequences vectors and host cells of the present invention forthe expression of genes with tight repression under basal i.e. noninducing conditions. As is shown in the present invention, theintroduction of additional repressor responsive elements not only givesan increase in expressed protein but also results in a lowertranscription rate under non inducing conditions. RT-PCR demonstratedthat the increase in secretion was the result of increased promoteractivity since higher concentrations of mRNA were present in theirradiated samples. Increased secretion of therapeutic proteins likemTNF-α in Clostridium after irradiation is thus the result of increasedactivity at transcriptional level. RT-PCR also demonstrated that innon-irradiated conditions, (resembling basal conditions) the addition ofa Cheo box resulted in lower transcription and the deletion of a Cheobox in higher transcription. These results prove that the Cheo boxfunctions as a repressor binding site which becomes free after DNAdamage, caused by, for example, ionising irradiation, leading to aremoval of repression and increased transcription.

Also genes with wild type promoters which are inducible by DNA damagingconditions or compounds can be modified by the addition of additionalrepressor binding elements from promoters which are inducible by DNAdamaging compounds or conditions, thereby further decreasing the basalexpression level of the genes which are operably linked to this modifiedpromoter when compared to the basal expression level of the unmodifiedpromoter.

This feature has several advantages for expression systems in general.For the expression of genes for proteins which are toxic to the host,the expression level under basal conditions has to be limited. Also forthe expression of genes which are introduced into host cells fortherapeutic agents the proteins preferably are not expressed under noninducing conditions.

Another embodiment relates to a novel in vitro expression system whereinhosts transfected with the vectors comprising the novel sequences of thepresent invention, wherein the protein expression is induced by DNAdamaging conditions or compounds. The in vitro expression system of thepresent can be used for the expression of any protein which is a likelycandidate to be expressed in bacterial cell. This expression system hasdesirable features such bulk growth of the host cells, inexpensive mediafor the growth of such host cells. In an embodiment of the presentinvention bacteria are exposed to radiation after a growth phase. Forexample, bacteria are exposed to 2 Gy with a ⁶⁰Cobalt unit at adose-rate of 0.9 Gy/min for a time period ranging from 1 to 120 minutesor 30 to 90 minutes.

In a preferred embodiment of the expression system the induction isperformed with UV irradiation. More preferably an incubator setting isused wherein the medium with host cells is transported along a UV lampwith a wavelength of 254 in order for a host cell to be subjected onaverage for about between 10 and 30 seconds, between 20 and 40 seconds,between 45 and 90 seconds, the distance between the host cell and the UVsource is between 1 and 5 cm or between 8 and 15 cm.

The introduction of repressor binding elements into a bacterial promoterin order to obtain higher expression, preferably with less basalexpression can be applied to modify any expression system which is knownthe person skilled in the art (for example, see Sambrook et al. citesupra, and vectors from commercial suppliers such as Novagen, Pharmacia,Gibco, Invitrogen, Stratagene)

Examples of constitutive promoters in E. coli which can be modifiedaccording to the present invention are the bla promoter fromβ-lactamase), the Tn5 promoter of the neomycin resistance gene, the catpromoter of the chloramphenicol resistance gene, the tet promoter of thetetracycline resistance gene, the strong constitutive EM7 and TRNApromoters.

EXAMPLE 1 Bacterial Strains, Plasmids and Culture Conditions

Clostridium acetobutylicum DSM792 was grown in 2×YT (Yeast Tryptone)medium at 37° C. in an anaerobic system (model 1024; Forma Scientific,Marietta, Ohio) with 90% N₂ and 10% H₂ and palladium as catalyst(Oultram et al. 1988, FEMS Micriobiol. Lett. 56, 83-88.

For primary vector construction, Escherichia coli TG1 was used (Sambrooket al cited supra). This strain was grown in Luria-Bertani (hereafterabbreviated as LB) broth at 37° C. E. coli strain ER2275 was used for invivo methylation of plasmid DNA prior to electroporation of clostridia(Mermelstein, L. D., and E. T. Papoutsakis. 1993. Appl. Environ.Microbiol. 59:1077-1081.; Mermelstein, L. D. et al, 1992. BioTechnology10:190-195.). The E.coli/Clostridium shuttle plasmid pIMP1 was used ascloning vector (Mermelstein, L. D. et al, 1992. BioTechnology10:190-195.).

The murine tumour necrosis factor alpha (hereafter abbreviated asmTNF-α) cDNA was available on plasmid pIG2mTNF (obtained fromInnogenetics, Gent, Belgium). Plasmid pHZ117, containing the eglA geneof C. acetobutylicum P262, was a obtained from H. Zappe (cited supra).The eglA promoter and signal sequence were used to express and secretemTNF-α. This chimeric gene construct was present on the shuttle plasmidpIMP1, resulting in pIMP-eglA-mTNF-α (Theys, J. et al, 1999, Appl.Environ. Microbiol. 65:4295-4300.). In this plasmid, the eglA promoterwas replaced by the C. acetobutylicum recA promoter, resulting inpIMP-recA-mTNF-α (Nuyts S. et al. 2001 Applied & EnvironmentalMicrobiology 67: 4464-4470.). Table 3 gives an overview of the plasmidsused in this study. The recA promoter was isolated from chromosomal DNAas previously described (Nuyts, S. et al, 2001, Radiat Res.155:716-726.). Media were supplemented, when applicable, witherythromycin (25 μg/ml) or ampicillin (50 μg/ml). TABLE 3characteristics of engineered plasmids Derived from Signal TherapeuticName plasmid Promoter sequence gene pIMP-eglA- pIMP1 eglA eglA mTNF-αmTNF-α pIMP- pIMP1 eglA with eglA mTNF-α eglACheo- incorporated mTNF-αCheo box pIMP-recA- pIMP1 recA eglA mTNF-α mTNF-α pIMP- pIMP1 recA witheglA mTNF-α recAextraCheo- extra Cheo mTNF-α box incorporated pIMP-pIMP1 recA with eglA mTNF-α recAdeletedCheo- Cheo box mTNF-α deleted

EXAMPLE 2 Mutation of the recA and eglA Promoter, DNA Manipulations andTransformation Procedures

Introduction and/or deletion of the Cheo box in the recA and eglApromoter was done using “Quickchange Site-directed Mutagenesis kit”(Stratagene). Table 4 represents the sequences of the ‘wild-type’ recAand eglA promoters at the 3′ region. All mutations were introduced inthe shuttle vectors pIMP1 containing the eglA or recA promoter followedby the eglA-mTNF-α fusion gene (see table 3). TABLE 4 Sequences of therecA and eglA promoter at the 3′ region. recA promoter [SEQ ID NO 54]

eglA promoter [SEQ ID NO 55]

-10 and -35 promoter elements are underlined. The Shine-Dalgarno (SD)sequence is boxed. Sequences being mutated in accordance with thepresent invention are underlined with interrupted line. The Cheo box ispresented in bold.

For mutation of the eglA and recA promoter, mutagenic primers containingan extra Cheo box flanked by 10-15 bases of the correct sequence were 5′TATATTGACAAATGAACAAATGTTCATATAATTATATG 3′ [SEQ ID NO44] and 5′CATATAATTATATGAACATTTGTTCATTTGTCAATATA 3′ [SEQ ID NO45] Primers todelete the Cheo box in the recA promoter region were 5′TAATTATATGTATA_(deletion 12 bp)GAGAGAAAGGTTGG 3′ [SEQ ID NO 46] and 5′CCAACCTTTCTCTC_(deletion 12 bp)TATACATATAATTA 3′ [SEQ ID NO 47] Primersto introduce a Cheo box in the eglA promoter region were 5′TTTAAGGGACTTTGAACATATGTTCTTGACAAATTAAT 3′ [SEQ ID NO 48] and 5′ATTAATTTGTCAAGAACATATGTTCAAAGTCCCTTAAA3′ [SEQ ID NO 49]. To verify theinsertion or deletion of the Cheo box, the DNA fragments containing theintroduced mutations were subcloned in pUC19 and the DNA sequence wasdetermined with an automated laser fluorescent ALF Express sequencer(Amersham Pharmacia BioTech). Primers used for sequencing were theCY5-labeled M13 forward and reverse primers.

All general DNA manipulations in E.coli were carried out as described bySambrook et al. (Sambrook, J. E. et al, 1989, Molecular cloning: alaboratory manual, 2^(nd) ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). Restriction endonucleases and DNA-modifyingenzymes were purchased from Roche Diagnostics (Brussels, Belgium), GIBCOBRL (Gaithersburg, Md.) and Eurogentec (Seraing, Belgium) and used asindicated by the suppliers. DNA plasmid isolations from E.coli wereperformed with the Wizard Plus SV miniprep kit (Promega Inc., Madison,Wis.).

E. coli was transformed using chemically competent cells obtained withthe RbCI method. Transformation of C. acetobutylicum DSM792 was carriedout by electroporation as published (Nakotte, S. et al, M., 1998, Appl.Microbiol. Biotechnol. 50:564-567).

EXAMPLE 3 Irradiation of Bacteria

Recombinant bacteria were grown until early log phase (OD_(600nm)=±0.3).At this time point cultures were divided into two sets, one of which wasexposed to radiation while the other was mock irradiated and used as acontrol. Bacteria were exposed to 2 Gy with a ⁶⁰Cobalt unit at adose-rate of 0.9 Gy/min. After irradiation, bacteria were incubatedanaerobically at 37° C. and samples were taken at different timeintervals after exposure. Each experiment was independently repeatedthree times.

EXAMPLE 4 Analysis of mTNF-α Secretion

The amount of mTNF-α secreted by recombinant clostridia was quantifiedusing ELISA. Supernatant taken from irradiated and non-irradiatedcultures was diluted 10-fold in phosphate buffered saline plus 7.5%bovine serum albumin and 100 μl aliquots were put in a 96-wellmicrotiter plate in duplicate. Further manipulations were done accordingto the manufacturer's protocol (DiaMed EuroGen, Tessenderlo, Belgium).

Concentrations of secreted mTNF-α were calculated and compared for theirradiated and non-irradiated cultures. The level of radio-inducedmTNF-α production was expressed as the relative increase in mTNF-αconcentration of irradiated samples compared with the correspondingnon-irradiated samples. Immunoblot analysis with polyclonal rabbitanti-mTNF-α antibodies was carried out by the method of Van Mellaert etal. (Van Mellaert, L. et al,. 1994, Gene 150:153-158.).

EXAMPLE 5 Reverse Transcriptase-PCR (RT-PCR)

To prove that induction of mTNF-α was the result of an increase inpromoter activity, RT-PCR was performed on RNA isolated from irradiatedand non-irradiated bacterial cultures. One hour after radiotherapy,aliquots of 4 ml culture were taken and RNA was extracted using the“RNeasy mini kit” from QIAGEN (Valencia, Calif.) as previously described(Nuyts, S., et al., 2001, J. Microbiol. Methods. 44:235-238.). RNAconcentration was determined spectrophotometrically. To avoid DNAcontamination which could result in mTNF-cDNA transcription from theplasmid, 1 μg RNA was digested with Fnu4HI which cleaves the mTNF-cDNAat position 82 and 213 and an additional DNase treatment was carriedout. After heat-inactivation of the enzymes, 200 U Murine-MoloneyLeukemia Virus-Reverse Transcriptase (M-MLV-RT) (GIBCO BRL,Gaithersburg, Md.) were added to the RNA together with 0.8 μl dNTPs (5mM each), 4 μl 5×RT-buffer, 2 μl reverse primer (10 pmol/μl) and 2 μlDithiothreitol (0.1 M) in a total volume of 20 μl. After 1 hourincubation at 37° C., the resulting cDNAs were amplified using PCR. 5 μlof the RT-mixture was added to 8.5 μl reverse primer, (10 pmol/μl), 7 μlforward primer (10 pmol/μl), 2.5 μl dNTPs (5 mM each), 0.5 U JumpStartTaq DNA Polymerase (Sigma, Sigma Chemical Co., St. Louis, Mo.) and 4.5μl 10×buffer in a total volume of 50 μl. After 40 PCR-cycles (10 min 95°C., 30 sec 95° C., 2 min 40° C., 30 sec 72° C., 5 min 72° C.) 1 μlaliquots were run on 1% agarose gels. Primers used for amplification ofmTNF-α were: Forward primer: 5′ GTAAGATCAAGTAGTCAA 3′ [SEQ ID NO 50] andReverse primer: 5′ CAGAGCAATGACTCCAAA 3′ [SEQ ID NO 51]. To verify theabsence of any DNA contamination all samples underwent the same RT-PCRreactions, without the addition of M-MLV-RT. To ensure equal amounts ofRNA in all samples, an internal fragment of Clostridium acetobutylicum16S rRNA was amplified using RT-PCR to function as internal standard.Primers used for amplification of 16S rRNA were Forward primer:5′GGAGCAAACAGGATTAGATACC 3′ and [SEQ ID NO 52] Reverse primer:5′ TGCCAACTCTATGGTGTGACG 3′. [SEQ ID NO 53]

EXAMPLE 6 Mutation of the recA and eglA Promoter

After introduction of mutations in the vectors pIMP-eglA-mTNF-α andpIMP-recA-mTNF-α by PCR mutagenesis, mutations were verified by sequenceanalysis and restriction digestion. Therefore, a 605 bp fragment of bothplasmids containing the mutated sequence were subcloned in pUC19digested with HindII.

Since both eglA and recA promoter are functional in E.coli, it waspossible to test activity of the mutated promoters by determination ofexpression and secretion of mTNF-α. Lysates and supernatants wereanalysed via Western blot analyses using rabbit anti-mTNF-α polyclonalantibodies and alkaline phosphatase conjugated anti-rabbit antibodies(Sigma, Sigma Chemical Co., St. Louis, Mo.). In both cell lysates andsupernatants clearly the presence of mTNF-α was demonstrated by allrecombinant bacteria containing the different constructs, proving thatthe mutated promoters were still functional.

After introduction of the recombinant plasmids into Clostridium viaelectroporation, presence of mTNF-α was again demonstrated insupernatants and lysates via immunoblotting.

EXAMPLE 7 Results on the Analysis of mTNF-α Secretion

ELISA analysis was used to quantify mTNF-α secretion by recombinantclostridia. As shown earlier, the ‘wild-type’ recA promoter gives a1.44-fold increase of mTNF-α secretion after a single dose of 2 Gy.After deletion of the Cheo box from the recA promoter region, nosignificant increase of mTNF-α secretion was measured after irradiationcompared with the control samples (FIG. 1). However, incorporation of anextra Cheo box in the recA promoter region, resulted in a 4.12-foldincrease in mTNF-α secretion 2.5 hrs after a single dose of 2 Gy (FIG.1). 1.5 hrs after radiation, the increase in mTNF-α secretion increasedby a factor of about 2.3.

Irradiation of the recombinant bacteria containing the pIMP-eglA-mTNFconstruct, resulted in no increase in mTNF-α secretion, confirming theconstitutive properties of the eglA promoter (FIG. 2). However, when aCheo box was incorporated in the eglA promoter region, a 2.42-foldincrease was seen 2.5 hrs after 2 Gy irradiation (FIG. 2). Again, 1.5hrs after radiation, the increase in mTNF-α secretion was 1.93.

The present example demonstrates that strong constitutive promoters suchas the eglA promoter can be made radio-inducible by introducing a Cheobox in the promoter region. This implies that secretion of high dosestherapeutic proteins like TNF-α can be controlled by ionisingirradiation. Since the Cheo box is functional in the eglA promoter,independently of its natural sequence context, it will be possible toradio-induce other clostridial promoters, which might even be stronger.Moreover the addition of more Cheo boxes will increase repression andhence augment inducibility further.

EXAMPLE 8 Results on the RT-PCR

Reverse transcriptase-PCR was carried out to prove that the increase inmTNF-α secretion was the result of an increase in promoter activity. 1μl of the PCR-mixture was put on gel (FIG. 3). The upper panelrepresents the 650 bp internal fragment of 16S rRNA, which was amplifiedto ensure equal amounts of RNA were used in each PCR reaction. The lowerpanel represents the 470 bp internal fragment of mTNF-α, which wasamplified. As shown the upper panel in FIG. 3, equal amounts of RNA wereused in all reactions. When mTNF-α was amplified, both for theconstructs with the eglA promoter with a Cheo box introduced (lanes 1and 2), the ‘wild-type’ recA promoter (lanes 3 and 4) and the recApromoter with the extra Cheo box (lanes 5 and 6), the samples fromnon-irradiated conditions result in a weaker band than the samplesirradiated, indicating that more mRNA was present in the irradiatedsamples. For the constitutive eglA promoter (lanes 8 and 9) and the recApromoter with a deletion of the Cheo box (lanes 10 and 11), nodifference can be seen between the irradiated and the non-irradiatedsamples. For the control samples, both the recA promoter with an extraCheo box and the eglA promoter containing a Cheo box, showed a weakerband than the corresponding ‘wild-type’ promoters. This weaker signal isattributed to lower transcription levels because of higher repressionlevels under non-induced conditions. The reverse is seen for the recApromoter with a deletion of the Cheo box: a higher signal in thenon-irradiated samples for the mutated promoter could be seen incomparison with the ‘wild-type’ promoter. This higher signal is theresult of the absence of repression.

This experiment shows that the addition of repressor responsive elementsresult lower basal expression levels.

The absence of any band in the samples to which no reverse transcriptasewas added, confirmed there was no DNA contamination present in none ofthe samples.

EXAMPLE 9 Radiation Induced Expression in Gram Negative Bacteria

Construction of the recombinant plasmid were carried out and thentransformed in E. coli TG1 (supE hsdΔ5 thi Δ(lac-proAB) F′ [traD36 proAB⁺ lacl^(q) lacZΔM15] strain. E.coli cells were routinely grown at 37°C. in LB medium under aerobic conditions by shaking. Chromosomal DNA wasextracted from TG1 E. coli cells using a wizard genomic DNA purificationkit (Promega). To isolate the recA promoter, PCR was carried out byusing this chromosomal DNA with specific oligonucleotides designed basedon the E coli DNA sequence with EMBL Accession number EC V00328,incorporating restriction sites (KpnI and Bg/II) at the 5′ end of eachprimers (ECRECA1 5′-TAGGTACCGTCTGGTTTGCTTGC-3′ [SEQ ID NO 56]; ECRECA25′-TAAGATCTCATGCCGGGTAATACC-3′ [SEQ ID NO 57]. DNA fragments of theexpected size were amplified and cloned into pGEM-T Easy vector(Promega) resulting into plasmid pRecA-GEM-T Easy. This plasmid wasdigested with KpnI and Bg/II to adapt the termini for in-frame insertionof the recA promoter into Kpn1-Bg/II sites in the pSp-luc+NF fusionvector (Promega). The resultant expression plasmid was designatedpRecA-Luc+NF (table 1). All restriction enzymes, T4 DNA ligase andpolymerase were from GIBCO BRL (Gaithzersburg, Md.), and Eurogentec(Seraing,Belgium) and used as indicated by the supplier. The conditionused for plasmid DNA extractions, restriction endonuclease digestion,agarose gel electrophoresis and isolation and ligation of DNA fragmentshave been carried out according to standard protocol (Sambrook, et al.1989). Plasmid DNA was isolated from E. coli with a Wizard Plus SVminiprep kit (Promega Inc, Madison, Wis.). Plasmid pRecA-GEM-T Easy wastransformed into chemically competent E. coli cells obtained with theRbCI method. Selection of the transformants carrying the appropriateplasmid was made on the bases of ampicillin resistance and Blue/whitecolony on the LB agar plate containing ampicillin and5-bromo-4chloro-3-indolyl-B-D-galactopyranoside (X-gal) and IPTG.Ampicillin, the X-gal and IPTG were used at a final concentration of 50,50 and 200 μg/ml respectively.

The sequence of cloned products was verified by automatic sequencingusing the thermo sequanase fluorescent labeled Amersham cycle sequencingkit based on Sanger dideoxy-method for sequencing in theALFexpress®Autoread® Sequencer (Amersham Pharmacia Biotech) and comparedwith the deposited sequence

Luciferase Reporter Assays: Cultures of transformed bacteria withpRecA-Luc+NF were grown to OD600 of about 0.3 (˜1×10.8.cells/ml).Subsequently, these cultures were divided into two fractions. Onefraction was irradiated with UV at a dose of 254 nm for 60 sec in aPetri-dish. The irradiated culture was reintroduced into the tube) andsimilarly as the non-irradiated culture continued to grow at 37° C. withshaking. The aliquot of the cultures were collected every 15 minutestill 1 hour followed with every 30 minute till 120 min., and theluciferase activity was determined.

20 μl aliquot of sample and 100 μl of luciferase assay reagent wereplace in black 96 well plate and placed in to luminometer chamber at atemperature control of 20° C. enzyme activity were measured using aPackard Lumicount micro plate luminometer. All measurements were takenat 0.5 and 2 sec per well-read length. Luminescence values are presentedas relative light units (RLU)(as per the particular instrument'soutput). The induction factor was calculated by dividing the enzymeactivity of an induced sample displayed at the times indicated by thatof a matched non-induced sample. The induction factor of 1.0 representsno induction. Each induction experiment was repeated three or more timesand the mean values are shown in table 5 and FIGS. 4 and 5. TABLE 5activity of a reporter gene under the control of a radiation induciblepromoter. Non Exposed Exposed non exposed exposed ((relative (relativelight (induction (induction Time (sec) light units units) factor)factor) 0 16200 19700 1 1.21 15 24900 86000 1 3.46 30 33700 119800 13.55 45 30800 184400 1 5.99 60 47800 195200 1 4.08 90 58900 188400 13.20 120 75700 190400 1 2.52

These results show that the radiation induced expression of aheterologous gene under the control of the RecA. promoter leads to afive fold higher relative induction than the basal expression under nonirradiated conditions.

The present example shows that the RecA promoter alone is sufficient topreform radiation induced expression outside its natural genomic DNAenvironment. Since the radiation induced expression in both Gramnegative bacteria and Gram positive bacteria is determined by therepressor binding elements.

1. An isolated and purified polynucleotide comprising at least one firstsequence element inserted in a second sequence element wherein the firstsequence element is a repressor binding element of a promoter which isinducible by DNA damaging agents or conditions and wherein the secondsequence element is a promoter sequence. 2-41. (cancelled).
 42. Thepolynucleotide of claim 1, wherein the promoter sequence of the secondsequence element is from a promoter which is not inducible by a DNAdamaging agent or condition.
 43. The polynucleotide of claim 1, whereinthe promoter sequence of the second sequence element is from a promoterwhich is inducible by a DNA damaging agent or condition.
 44. Thepolynucleotide of claim 1, wherein said polynucleotide is positioned 5′to a nucleotide sequence suitable for the introduction of a thirdsequence element.
 45. The polynucleotide of claim 1, wherein theinsertion of a first sequence element occurs between about 46 base pairsand about 106 base pairs upstream of the ribosome binding site of thesecond sequence element.
 46. The polynucleotide of claim 42, wherein thenon-inducible promoter is a constitutive promoter or an induciblepromoter.
 47. The polynucleotide of claim 42, wherein the non-induciblepromoter is a bacterial promoter.
 48. The polynucleotide of claim 47,wherein the bacterial promoter is from gram positive bacteria.
 49. Thepolynucleotide of claim 47, wherein the bacterial promoter is from gramnegative bacteria.
 50. The polynucleotide of claim 47, wherein thebacterial promoter is an EglA promoter of Clostridium sp.
 51. Thepolynucleotide of claim 43, wherein the inducible promoter is abacterial promoter.
 52. The polynucleotide of claim 51, wherein thebacterial promoter is from gram positive bacteria.
 53. Thepolynucleotide of claim 51, wherein the bacterial promoter is from gramnegative bacteria.
 54. The polynucleotide of claim 53, wherein thebacterial promoter is RecA.
 55. The polynucleotide of claim 1, whereinthe repressor binding element comprises a Cheo box consensus sequence asdepicted in SEQ ID NO:
 1. 56. The polynucleotide of claim 1, wherein therepressor binding element comprises a DinR box consensus sequence asdepicted in SEQ ID NO:
 2. 57. The polynucleotide of claim 1, wherein therepressor binding element comprises a sequence selected from the groupof sequences depicted in SEQ ID 4 to SEQ ID
 25. 58. The polynucleotideof claim 1, wherein the repressor binding element comprises a SOS boxconsensus sequence as depicted in SEQ ID NO3.
 59. The polynucleotide ofclaim 1, wherein the repressor binding element comprises a sequenceselected from the group of sequences depicted in SEQ ID 26 to SEQ ID 43.60. The polynucleotide of claim 44, wherein the third sequence elementencodes a protein with pharmaceutical properties or a protein which isable to convert an inactive compound into a pharmaceutically activecompound.
 61. The polynucleotide according to claim 60 wherein theprotein with therapeutic properties is TNF-alpha (Tumour Necrosis Factoralpha).
 62. A method of converting a promoter which is not inducible byDNA damaging agents or conditions into a promoter which is inducible byradiation, genotoxic compounds or DNA damaging compounds comprising thestep of inserting at least one repressor binding element of a promoterwhich is inducible by a DNA damaging compound or condition into said noninducible promoter.
 63. A method of increasing the induction level of afirst promoter which is inducible by genotoxic compounds or conditionscomprising the step of inserting at least one repressor binding elementof said first promoter or of a second promoter which is inducible by aDNA damaging compound or condition into said first inducible promoter.64. A method of decreasing the basal expression level of a firstpromoter which is inducible by genotoxic compounds or conditionscomprising the step of inserting at least one repressor binding elementof said first promoter or of a second promoter which is inducible by aDNA damaging compound or condition into said first inducible promoter.65. A vector comprising a nucleotide sequence according to claim
 1. 66.A bacterial host cell transfected with the vector of claim
 65. 67. Abacterial host cell according to claim 66, wherein said cell is afacultative or obligate anaerobic bacterium.
 68. A pharmaceuticalcomposition comprising a cell according to claim 66 in admixture with atleast one pharmaceutically acceptable carrier.
 69. A pharmaceuticalcomposition comprising a cell according to claim 67 in admixture with atleast one pharmaceutically acceptable carrier.
 70. A method for the invitro production of recombinant proteins comprising the step ofcontacting a culture of host cells according to claim 66 with a DNAdamaging compound or condition.